Final Report Summary - NEURONAL POLARITY (Pyramidal neuron polarity decisions during migration and axonal outgrowth in the developing mammalian cerebral cortex)
The mammalian cerebral cortex is populated by two main types of neurons: the excitatory neurons and the inhibitory neurons. The excitatory type account for the majority of neurons found in the cortex. During embryonic life, the development of the brain requires that neurons migrate away from their birth place in order to perform their functions properly. In addition, neurons have to extend neurites and ultimately differentiate and communicate with each other. Characterisation of the molecular signalling pathways involved in cerebral cortex development is important for the understanding of brain pathologies such as lissencephaly, microcephaly, periventricular heterotopia, epilepsy, dyslexia, mental retardation, schizophrenia, bipolar disorder, and many others resulting from defective cortical architecture, connectivity and function.
In our study, we showed that the neuron-specific inhibition of the small GTPase Rap1 (i.e. without affecting other cell type also present in the brain) in vivo induces an ectopic accumulation of neurons halfway towards their final position. Time lapse video-microscopy and immunostaining for polarity markers revealed that this phenotype is not the result of defective neuronal motility of the affected cells, but is rather due to a defect in their polarisation. This is because the movement of the Rap-inhibited neurons is randomised with a decreased net movement towards the correct direction. The absence of effect on motility has been confirmed in an in vitro lattice culture system where dissociated neurons move along glial fibres. The in vivo phenotype does not show a block of all the affected cells. Indeed, some of them migrate all the way and reach their final position, albeit with a significant delay when compared to control cells. Together, these observations suggest that Rap is important for the initial polarisation of neurons but not migration per se.
Our recent findings also demonstrated that in the mammalian cerebral cortex the transmembrane receptor N-cadherin (NCad) has an important function in polarising cortical neurons. The inhibition of cadherins in neurons without affecting progenitor cells and their radial glia fibers, recapitulates the phenotype induced by inhibition of Rap1. Several experiments confirmed that NCad functions downstream from Rap1. First, inhibition of Rap1 in vivo and in vitro reduced the presence of NCad at the plasma membrane with a concomitant increase in intracellular NCad. Second, a functional assay demonstrated that inhibition of Rap1 reduced the binding of neurons to the NCad extracellular domain. And finally, over-expression of NCad in the cortex is able to partially rescue the cell positioning defect due to inhibition of Rap1. These data suggest that Rap1 activity is important in migrating neurons in order to maintain the high level of NCad at the plasma membrane necessary to allow cells to polarise correctly. Yet we do not know whether other cadherins, also expressed in the cortex, might have some redundant function with NCad. In addition, how NCad allows the polarisation of cortical neurons is still under investigation. Nevertheless, hypotheses might be suggested. NCad may be activated locally in order to increase the binding to radially-oriented processes on other neurons or glial fibres. This adhesion could stabilise the position of the centrosome. However, in other cell types, it is the cadherin-free cell edge that shows the polarity of migration. Indeed, cadherin-mediated cell-cell interactions induce the centrosome and Golgi apparatus to move towards the free cell edges in cultured astrocytes and stimulate protrusions at the free edge in Xenopus neural crest cells. Alternatively, NCad may be a regulator for other cell surface receptors that respond to directional signals.
In our study, we showed that the neuron-specific inhibition of the small GTPase Rap1 (i.e. without affecting other cell type also present in the brain) in vivo induces an ectopic accumulation of neurons halfway towards their final position. Time lapse video-microscopy and immunostaining for polarity markers revealed that this phenotype is not the result of defective neuronal motility of the affected cells, but is rather due to a defect in their polarisation. This is because the movement of the Rap-inhibited neurons is randomised with a decreased net movement towards the correct direction. The absence of effect on motility has been confirmed in an in vitro lattice culture system where dissociated neurons move along glial fibres. The in vivo phenotype does not show a block of all the affected cells. Indeed, some of them migrate all the way and reach their final position, albeit with a significant delay when compared to control cells. Together, these observations suggest that Rap is important for the initial polarisation of neurons but not migration per se.
Our recent findings also demonstrated that in the mammalian cerebral cortex the transmembrane receptor N-cadherin (NCad) has an important function in polarising cortical neurons. The inhibition of cadherins in neurons without affecting progenitor cells and their radial glia fibers, recapitulates the phenotype induced by inhibition of Rap1. Several experiments confirmed that NCad functions downstream from Rap1. First, inhibition of Rap1 in vivo and in vitro reduced the presence of NCad at the plasma membrane with a concomitant increase in intracellular NCad. Second, a functional assay demonstrated that inhibition of Rap1 reduced the binding of neurons to the NCad extracellular domain. And finally, over-expression of NCad in the cortex is able to partially rescue the cell positioning defect due to inhibition of Rap1. These data suggest that Rap1 activity is important in migrating neurons in order to maintain the high level of NCad at the plasma membrane necessary to allow cells to polarise correctly. Yet we do not know whether other cadherins, also expressed in the cortex, might have some redundant function with NCad. In addition, how NCad allows the polarisation of cortical neurons is still under investigation. Nevertheless, hypotheses might be suggested. NCad may be activated locally in order to increase the binding to radially-oriented processes on other neurons or glial fibres. This adhesion could stabilise the position of the centrosome. However, in other cell types, it is the cadherin-free cell edge that shows the polarity of migration. Indeed, cadherin-mediated cell-cell interactions induce the centrosome and Golgi apparatus to move towards the free cell edges in cultured astrocytes and stimulate protrusions at the free edge in Xenopus neural crest cells. Alternatively, NCad may be a regulator for other cell surface receptors that respond to directional signals.